Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci

Abstract

Analysis of a region involved in the conjugative transfer of the lactococcal conjugative element pRS01 has revealed a bacterial group II intron. Splicing of this lactococcal intron (designated Ll.ltrB) in vivo resulted in the ligation of two exon messages (ltrBE1 and ltrBE2) which encoded a putative conjugative relaxase essential for the transfer of pRS01. Like many group II introns, the Ll.ltrB intron possessed an open reading frame (ltrA) with homology to reverse transcriptases. Remarkably, sequence analysis of ltrA suggested a greater similarity to open reading frames encoded by eukaryotic mitochondrial group II introns than to those identified to date from other bacteria. Several insertional mutations within ltrA resulted in plasmids exhibiting a conjugative transfer-deficient phenotype. These results provide the first direct evidence for splicing of a prokaryotic group II intron in vivo and suggest that conjugative transfer is a mechanism for group II intron dissemination in bacteria.Conjugation is an important mode of genetic exchange in bacteria. The specific mechanism of DNA transfer comprises two distinct functions, one being the enzymatic preparation of the plasmid DNA prior to replicative transfer and another involving formation of the mating channel through which DNA is transferred into a recipient cell. Initiation of plasmid transfer requires a single-stranded cleavage at a specific origin of transfer (oriT) produced by the action of a specialized nucleoprotein complex called a relaxosome (53). In many conjugative systems, transfer origins are flanked by genes involved in the formation of a functional relaxosome complex. One key feature of an oriT region is the ability of the region to confer mobilization in cis, provided the remaining transfer functions are present in trans.The conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 and the sex factor from L. lactis subsp. lactis 712 are prototypical mobile elements in lactococci (15, 50). Both elements have been shown to mediate high-frequency transfer of genes encoding lactose utilization (Lac ϩ ) by insertion sequence-directed conintegration with nonconjugative Lac ϩ plasmids (19,40). In addition, both elements confer a cell aggregation (Clu) phenotype (16, 52) associated with highfrequency conjugative transfer. Previous genetic analysis of these elements has identified the gene responsible for the aggregation phenotype (clu) associated within an inversion region (2, 20, 21). To date, however, the oriT and gene(s) encoding relaxosome components of pRS01 have not been localized within either element. In an effort to exploit conjugation as a means of lactococcal strain development, we have characterized the transfer regions of pRS01 by insertional mutagenesis via IS946-mediated cointegration with the 11-kb plasmid pTRK28 (43) (for a representation of pTRK28:: pRS01 cointegration, see Fig. 1 in reference 33). Analysis of the insertion site junctions of pRS01::pTRK28 cointegrate plasmids identified four distinct regions of pRS01 involved...

Trans-splicing versatility of the Ll.LtrB group II intron

“…The maturase activity of LtrA promotes intron splicing by stabilizing the catalytically active tertiary conformation of the ribozyme (Matsuura et al 1997). Ll.LtrB was found on two highly similar conjugative elements of L. lactis: a conjugative plasmid, pRS01 (Mills et al 1996), and a chromosomal sex factor (Shearman et al 1996). In both elements, Ll.LtrB interrupts the ltrB gene coding for relaxase (LtrB) at the same position.…”

“…This enzyme is thus essential for conjugation, and splicing of Ll.LtrB from its pre-mRNA transcript is necessary for LtrB production and subsequent DNA transfer. Therefore, Ll.LtrB splicing controls conjugation efficiency of its host elements (Mills et al 1996;Shearman et al 1996). We previously developed a sensitive splicing/conjugation assay in L. lactis to show that Ll.LtrB trans-splices when fragmented at natural group II intron fragmentation sites.…”

Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

“…1), predicted to encode a relaxase protein, is most closely related (at both the DNA and protein sequence levels) to the ItrB gene of the lactococcal conjugative element pRS01 (Mills et al, 1994). An interesting feature of ltrB is that the wild-type allele is interrupted by the self-splicing, mobile group II intron Ll.ltrB, the first fully functional group II intron identified in bacteria (Mills et al, 1996). We recently showed that Ll.ltrB efficiently targets DNA of a conserved relaxase domain in pcfG for insertion by a retro-transposition mechanism (Staddon et al, 2004).…”

Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron

“…The Ll.LtrB group II intron from the low G+C grampositive bacterium Lactococcus lactis interrupts a putative relaxase gene (ltrB), which is present on three conjugative elements: the pRS01 (48.4 kb) (Mills et al 1996) and pAH90 (26.5 kb) (O'Sullivan et al 2001) plasmids, and an integrative and conjugative element called the sex factor (50 kb) (Shearman et al 1996). L. lactis is a mesophilic lactic acid bacterium (LAB) that is widely used in the dairy industry, for instance, in the manufacturing of cheese.…”

“…Ll.LtrB was the first bacterial group II intron that was shown to splice (Mills et al 1996;Shearman et al 1996) and invade new sites in vivo (Mills et al 1997). The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in L. lactis and Escherichia coli (Cousineau et al 1998(Cousineau et al , 2000Ichiyanagi et al 2002Ichiyanagi et al , 2003Coros et al 2005;Smith et al 2005).…”